The Crystal Structure of<i>Bacillus cereus</i>Phosphonoacetaldehyde Hydrolase:  Insight into Catalysis of Phosphorus Bond Cleavage and Catalytic Diversification within the HAD Enzyme Superfamily<sup>,</sup>

Morais, Marc C.; Zhang, Wenhai; Baker, Angela; Zhang, Guofeng; Dunaway‐Mariano, Debra; Allen, Karen N.

doi:10.1021/bi001171j

Cited by 138 publications

(176 citation statements)

References 47 publications

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“…Figure 2 shows the superposition of the backbones of these three HAD family phosphotransferases. The average B-factor of the -PGM cap domain is higher (B factor ) 41.9) than that of the core domain (B factor ) 26.58), consistent with the known mobility of the cap domain in several HAD structures (4,10,11).…”

Section: Resultssupporting

confidence: 78%

“…Superposition of the two structures reveals a conserved active-site platform within the core domain comprised of four peptide loops (10) (see Figure 5 for superposition (a) and schematic (b)). These four loops correspond to the four conserved sequence motifs within the HAD enzyme family which had been noted previously by us (10,26) and by other investigators (4,5,24,27,28). The structures of 2-haloalkanoic acid dehalogenase (4), phosphonatase (10), Ca 2+ -ATPase (29), phosphoserine phos- We are most intrigued by how the four phosphotransferases have become specialized to perform their specific catalytic function.…”

Section: Resultsmentioning

confidence: 74%

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Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

et al. 2002

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Phosphoglucomutases catalyze the interconversion of D-glucose 1-phosphate and D-glucose 6-phosphate, a reaction central to energy metabolism in all cells and to the synthesis of cell wall polysaccharides in bacterial cells. Two classes of phosphoglucomutases (R-PGM and -PGM) are distinguished on the basis of their specificity for R-and -glucose-1-phosphate. -PGM is a member of the haloacid dehalogenase (HAD) superfamily, which includes the sarcoplasmic Ca 2+ -ATPase, phosphomannomutase, and phosphoserine phosphatase. -PGM is unusual among family members in that the common phosphoenzyme intermediate exists as a stable ground-state complex in this enzyme. Herein we report, for the first time, the three-dimensional structure of a -PGM and the first view of the true phosphoenzyme intermediate in the HAD superfamily. The crystal structure of the Mg(II) complex of phosphorylated -phosphoglucomutase ( -PGM) from Lactococcus lactis has been determined to 2.3 Å resolution by multiwavelength anomalous diffraction (MAD) phasing on selenomethionine, and refined to an R cryst ) 0.24 and R free ) 0.28. The active site of -PGM is located between the core and the cap domain and is freely solvent accessible. The residues within a 6 Å radius of the phosphorylated Asp8 include Asp10, Thr16, Ser114, Lys145, Glu169, and Asp170. The cofactor Mg 2+ is liganded with octahedral coordination geometry by the carboxylate side chains of Asp8, Glu169, Asp170, and the backbone carbonyl oxygen of Asp10 along with one oxygen from the Asp8-phosphoryl group and one water ligand. The phosphate group of the phosphoaspartyl residue, Asp8, interacts with the side chains of Ser114 and Lys145. The absence of a base residue near the aspartyl phosphate group accounts for the persistence of the phosphorylated enzyme under physiological conditions. Substrate docking shows that glucose-6-P can bind to the active site of phosphorylated -PGM in such a way as to position the C(1)OH near the phosphoryl group of the phosphorylated Asp8 and the C(6) phosphoryl group near the carboxylate group of Asp10. This result suggests a novel two-base mechanism for phosphoryl group transfer in a phosphorylated sugar.In this paper, we report the three-dimensional structure of the -phosphoglucomutase from Lactococcus lactis. Both R-and -phosphoglucomutases catalyze the interconversion of D-glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P). This reaction is central to energy metabolism in all cells and is essential to the synthesis of cell wall polysaccharides in bacterial cells (1, 2). The R-phosphoglucomutase (R-PGM) acts on the R-C(1) anomer of G1P, while the -phosphoglucomutase ( -PGM) catalyzes the reaction of the -C(1) anomer. Both mutases employ Mg 2+ and -or R-glucose 1,6-diphosphate (G1,6-diP) as cofactors (1, 2). In addition, both mutases are monomeric proteins. The R-PGM (65 kDa) is, however, approximately twice the size of the -PGM (25 kDa) (3). The X-ray structure of R-PGM from rat reveals a 4-domain R-/ -protein. All four domains contribute residue...

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Section: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 74%

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

et al. 2002

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“…Analysis of the refined final structure illustrated the same overall fold as that of the wild-type phosphonatase ( Figure 7a) made of a core and a cap domain (1). The wild-type quaternary interactions between homodimers are maintained in the mutant structure.…”

Section: Kinetic Properties Ofmentioning

confidence: 74%

“…The mutant was crystallized in the presence of Mg 2+ , sulfate (from the crystallization conditions), and vso 3 (a competitive inhibitor with inhibition constant, K i ) 180 ( 20 µM). The structure was determined to 2.8 Å resolution by molecular replacement using the wild-type phosphonatase-Mg 2+ -tungstate structure (1).…”

Section: Kinetic Properties Ofmentioning

confidence: 99%

Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain^,

et al. 2004

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The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the R/ core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight -turn in the helix-loophelix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and -phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.

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“…The Mg(II) bound in this structure (7), -D-glucose 1,6-(bis)phosphate bound to -PGM (PDB entry 1O03) (30), the WO 4 2--bound structure of phosphonatase (PDB entry 1FEZ) (6), and the phosphate-bound structure of human mitochondrial deoxyribonucleotidase (PDB entry 1MH9) (31), a similar arrangement is observed with one ligand from the substrate, two ligands from loop I (the catalytic aspartate carboxylate group and the backbone carbonyl oxygen two residues downstream), and three from aspartates in loop IV (either directly or though water). The variability is in one of these water ligands to Mg(II).…”

Section: Mg(ii) and Phosphate-binding Residues Of The Active Sitementioning

confidence: 99%

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily^,

et al. 2004

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The haloacid dehalogenase (HAD) superfamily is comprised of structurally homologous enzymes that share several conserved sequence motifs (loops I-IV) in their active site. The majority of HAD members are phosphohydrolases and may be divided into three subclasses depending on domain organization. In classes I and II, a mobile "cap" domain reorients upon substrate binding, closing the active site to bulk solvent. Members of the third class lack this additional domain. Herein, we report the 1.9 Å X-ray crystal structures of a member of the third subclass, magnesium-dependent phosphatase-1 (MDP-1) both in its unliganded form and with the product analogue, tungstate, bound to the active site. The secondary structure of MDP-1 is similar to that of the "core" domain of other type I and type II HAD members with the addition of a small, 28-amino acid insert that does not close down to exclude bulk solvent in the presence of ligand. In addition, the monomeric oligomeric state of MDP-1 does not allow the participation of a second subunit in the formation and solvent protection of the active site. The binding sites for the phosphate portion of the substrate and Mg(II) cofactor are also similar to those of other HAD members, with all previously observed contacts conserved. Unlike other subclass III HAD members, MDP-1 appears to be equally able to dephosphorylate phosphotyrosine and closed-ring phosphosugars. Modeling of possible substrates in the active site of MDP-1 reveals very few potential interactions with the substrate leaving group. The mapping of conserved residues in sequences of MDP-1 from different eukaryotic organisms reveals that they colocalize to a large region on the surface of the protein outside the active site. This observation combined with the modeling studies suggests that the target of MDP-1 is most likely a phosphotyrosine in an unknown protein rather than a small sugar-based substrate.

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The Crystal Structure ofBacillus cereusPhosphonoacetaldehyde Hydrolase: Insight into Catalysis of Phosphorus Bond Cleavage and Catalytic Diversification within the HAD Enzyme Superfamily^,

Cited by 138 publications

References 47 publications

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain^,

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily^,

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The Crystal Structure ofBacillus cereusPhosphonoacetaldehyde Hydrolase: Insight into Catalysis of Phosphorus Bond Cleavage and Catalytic Diversification within the HAD Enzyme Superfamily,

Cited by 138 publications

References 47 publications

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis,

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis,

Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain,

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily,

Contact Info

Product

Resources

About

The Crystal Structure ofBacillus cereusPhosphonoacetaldehyde Hydrolase: Insight into Catalysis of Phosphorus Bond Cleavage and Catalytic Diversification within the HAD Enzyme Superfamily^,

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain^,

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily^,